Brain-Targeted Biomimetic Nanodecoys with Neuroprotective Effects for Precise Therapy of Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the gradual loss of dopaminergic neurons in the substantia nigra and the accumulation of α-synuclein aggregates called Lewy bodies. Here, nanodecoys were designed from a rabies virus polypeptide with a 29 amino acid (RVG29)-modified red blood cell membrane (RBCm) to encapsulate curcumin nanocrystals (Cur-NCs), which could effectively protect dopaminergic neurons. The RVG29-RBCm/Cur-NCs nanodecoys effectively escaped from reticuloendothelial system (RES) uptake, enabled prolonged blood circulation, and enhanced blood–brain barrier (BBB) crossing after systemic administration. Cur-NCs loaded inside the nanodecoys exhibited the recovery of dopamine levels, inhibition of α-synuclein aggregation, and reversal of mitochondrial dysfunction in PD mice. These findings indicate the promising potential of biomimetic nanodecoys in treating PD and other neurodegenerative diseases.

S3 relative humidity. The mice were maintained on a 12-h light/dark cycle and received food and water ad libitum. All procedures involving experimental animals complied with the relevant guidelines provided by the Animal Ethics Committee of Guangzhou University of Chinese Medicine (NO. 20200529002).

Preparation and characterization of Cur-NCs
Cur-NCs were prepared using the anti-solvent precipitation method. 1 Cur powder was dissolved in acetone to prepare an organic phase with a concentration of 20 mg/mL. An aqueous phase was prepared with PVP K90 with a chosen concentration of 0.8 mg/mL. The organic phase was rapidly injected into the aqueous phase at a volume ratio of 1:50 and stirred at 1000 rpm/min to obtain Cur-NCs. The thermal properties of the Cur-NCs were measured using DSC (Netzsch DSC 200 F3), and the crystalline state of the Cur-NCs was analyzed using XRD at room temperature with a Brucker D8 X-ray diffractometer.

Molecular dynamics simulation
The PVP polymer (20 units per polymer block) and Cur molecular topology files were built based on the gaff force field, and the above system was placed in a cube box using GMX editconf. All molecular dynamics simulations were carried out using GROMACS 2018.4 software; the Amer99SB force field was selected, and the TIP3P model was used for water molecules. The Verlet leapfrog algorithm was used to solve the Newton equation of motion, and the integration step was set to 2 fs. In the calculation process, the van der Waals force was calculated based on the Lennard Jones function, and the non-bond truncation distance was set to 1.2 nm. The bond length of all atoms was constrained by the LinCS algorithm, and the long-range electrostatic interaction was calculated using the particle mesh Ewald method. The lattice width was set to 0.16 nm. Periodic boundary conditions were used in the simulation, and the molecular dynamics simulation period was 50 ns.

RBCm derivation
RBCms were collected based on a modified version of a previously reported method. 2 Briefly, fresh heparinized whole blood was collected from C57BL/6 mice and then centrifuged at 800 ×g at 4°C for 20 min to remove the plasma and buffy coat.
The RBCs obtained were washed thrice with 1× PBS and suspended for 1 h in 0.25× PBS in an ice bath (inverted every 10 min). Subsequently, the cracked RBCs were centrifuged at 9000 ×g at 4°C for 10 min to remove hemoglobin. Further, a hypotonic 0.25× PBS solution (over 4 times the volume) was added, and the RBCm was washed repeatedly until the supernatant was colorless. The collected pink RBCm was purified and stored in 1× PBS.

Synthesis of DSPE-PEG2000-RVG29
DSPE-PEG2000-MAL (200 mg) was dissolved in 5 mL N,N-dimethylformamide (DMF). Then, RVG29 polypeptide was added and dissolved completely, and the mixture was stirred at room temperature for 12 h. Then, the reaction solution was transferred to a dialysis bag (retaining molecular weight of 3500 kDa), dialyzed, and purified in pure water for 24 h. The dialysate was collected and further freeze-dried to obtain DSPE-PEG2000-RVG29. The synthesis of DSPE-PEG2000-RVG29 was verified using 1 H-NMR spectroscopy.

Preparation of RVG29-RBCm/Cur-NCs
The RBCms derived from 400 μL of blood were incubated with 100 μg/mL of DSPE-PEG2000-RVG29 (dispersed in PBS) in a 37°C water bath with shaking (4 h) to generate RVG29-inserted RBCm. To identify the optimal ratio of RBCm to Cur-NCs, the volume of RBCms derived from 400 μL of blood was set as 1 and then mixed with 10, 8, 6, and 4 mL of Cur-NCs separately. The RBCm and Cur-NCs were sonicated at 60 kHz for 8 min and then extruded through 400-nm, 200-nm, and 100-nm polycarbonate membranes 11 times using a lipid extruder to obtain RBCm/Cur-NCs.
The mixtures with different RBCm/Cur-NCs ratios were then incubated with 20% S5 FBS at 37°C for 3 h. The aggregation of particles was evaluated by measuring the absorbance at 560 nm. The zeta potential and particle size of RVG29-RBCm/Cur-NCs were obtained using a particle sizing system (PSS NICOMP 380ZLS, USA), and the morphology was observed using transmission electron microscopy (TEM).

In vitro release
Dialysis was employed to assess the in vitro release of Cur. Briefly, 1 mL of different Cur formulations was added to the dialysis bag (MWCO: 3500 kDa), which was then immersed in 10 mL of release external solution (PBS buffer, pH = 7.4, containing 0.5% Tween 80, v/v). This apparatus was placed in a 37 ± 0.5°C water bath under horizontal shaking at 100 rpm. The external solution was collected at 0, 1, 2, 4, 8, 10, 12, 24, and 48 h, and then supplemented with an equal volume of fresh medium.
The Cur content was detected using a UV spectrophotometer at 427 nm.

Identification of membrane protein retention
SDS-PAGE was employed to analyze the proteins present on the prepared nanoparticles.
RBCm, RVG29-RBCm, RBCm/Cur-NCs, and RVG29-RBCm/Cur-NCs were individually added to 4 times the volume of RIPA lysis buffer, shaken, and lysed for 30 min in a 4°C ice bath. The supernatant was collected after centrifugation at 10000 rpm. Then, 30 μg protein was added to each well of a 10% SDS gel and electrophoresis was performed at 100 V for 2 h. Finally, the gel was stained with Coomassie blue for 1 h and observed. Hank's buffer. The Cur concentration was detected using a UV-Vis spectrometer, and the apparent permeability coefficient (Papp) was calculated as follows:

In vitro BBB model establishment and evaluation of NC transport
where V is the volume (mL) of Hank's buffer in the receptor chamber, dC/dt is the amount of Cur permeating over time (ng/mL·s), A is the surface area (cm 2 ) of the filter, and C0 is the initial concentration (ng/mL) on the apical side.

Cytotoxicity assay
The and RAW264.7 cells was observed using CLSM.

Mechanism underlying the cellular uptake of RVG29-RBCm/Cur-NCs in bEnd.3 Cells
To understand the mechanism via which bEnd.3 cells take up RVG29-RBCm/Cur-NCs, the cells were pre-treated with inhibitors of specific S7 endocytosis pathway (Table S1) In vitro neuroprotective effect SH-SY5Y cells were initially plated at a density of 8 × 10 3 /well in 96-well plates.
Then, cells were incubated with MPP + (final concentration, 2 mM) for 24 h. Cell viability was measured using an MTT assay. Live/dead cell imaging was performed using calcein-AM (5 μg/mL) and PI (10 μg/mL) staining after different treatments.

Flow cytometry analysis
SH-SY5Y cells were plated at a density of 1.2 × 10 5 /well in 12-well plates. After S8 RVG29-RBCm/Cur-NCs for 2 h. Then, MPP + was added (final concentration, 2 mM) and the cells were incubated for 24 h. The cells were harvested and stained with Annexin V-FITC/PI for analyzing cell apoptosis. To detect intracellular ROS, the cells were treated with 10 μM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and quantified using flow cytometry.

Detection of mitochondrial membrane potential
SH-SY5Y cells were treated with Cur formulations and MPP + as described above.
Then, the cells were collected and stained using the JC-1 method. The red fluorescence of JC-1 aggregates (Ex/Em: 585/590 nm) and green fluorescence of JC-1 monomers (Ex/Em: 514/529 nm) were observed under a fluorescence microscope.

Immunofluorescence assay for α-syn
SH-SY5Y cells were first plated at a density of 1.2 × 10 5 /well in 12-well plates.
After 24 h, the cells were fixed and treated with a blocking solution (containing 0.25% Triton X-100 and 5% BSA) for 30 min. Next, the cells were incubated with a primary antibody against α-syn (1:100, Abcam) at 4°C overnight. Subsequently, these cells were incubated with a secondary antibody (1:500, Abcam) at room temperature for 2 h. At the end of this incubation, the cells were stained with DAPI, and α-syn staining was observed using CLSM.

Hemolysis test
Briefly, 150 μL of a purified 4% red blood cell suspension derived from

In vivo imaging
To evaluate the brain-targeting ability of the biomimetic NCs, the prepared nano-formulations were labeled with Cy5 via lipid insertion. Cy5, Cy5@RBCm/Cur-NCs, and Cy5@RVG29-RBCm/Cur-NCs were separately injected into the tail vein of healthy mice, and fluorescence signals were recorded from major organs (brain, heart, liver, spleen, lungs, and kidneys) at different time-points using a live imaging system (Berthold LB983). Fluorescence signals were then quantified S10 using IndiGo software.

Behavioral tests
For the pole test, the mice were placed in a head upward position on top of a vertical rod with a rough surface (1 cm in diameter and 50 cm in height). The mice spent on turning back (T-turn) and the total time required to climb to the bottom of the rod (T-total) were recorded. 6 For the rotarod test, the mice were placed on a rod (7 cm in diameter) rotating at a fixed speed of 30 rpm. During the 2-min test, the latency to fall and the number of drops were recorded for each animal. These experiments were repeated three times for each mouse, and analyses were performed using the average value.
For the open-field test, mice were individually placed in an empty box (60 cm × 60 cm × 40 cm), and the environment was kept dark and quiet during the experiment.
Before the test, each mouse was placed in the center of the empty box and allowed to explore it freely for 10 minutes to ensure adaption. Then, the trajectory of the mice was recorded for 15 min during the test and analyzed.
For the gait dynamics test, a Digigait TM Imaging system (Mouse Specifics Inc.) S11 was conducted as previously reported. 7 Firstly, the mice were trained to walk on the belt at a speed of 22 cm/s. Then, the gait of the mice walking continuously in the center of the visual field was recorded. The portions of the paw, gait parameters, and relevant index values were identified by the software. The definition of the parameters of interest are depicted in Table S3.

Quantification of TH + neurons
Immunofluorescence analysis was performed to quantify the number of TH + neurons in the SNpc according to previously described protocols. 8 . Brains harvested from mice were fixed in 4% paraformaldehyde (PFA) and then dehydrated in 30% sucrose. After dehydration, the brains were cut into 30-μm-thick sections, which were blocked with 10% goat serum for 30 min. The sections were then incubated with an anti-TH antibody (1:2000) overnight at 4°C. Subsequently, the sections were rinsed thrice in PBS and incubated with an Alexa fluor 594-conjugated secondary antibody at room temperature for 2 h. After DAPI staining, red fluorescence was observed under a fluorescence microscope, and the TH + cells in the SNpc were quantified using ImageJ software.

Detection of oxidative stress levels
The levels of SOD, malondialdehyde (MOD), and GSH in the striatum of mice were detected following standard protocols of Beyotime Biotechnology (Shanghai, China). The levels of glutamate dehydrogenase (GDH), ATP, ROS, and MOD in the midbrain were measured in a similar manner.

Measurement of DA levels
The striatum was isolated and weighed for further analysis. The sample was placed in 0.4 M/L HClO4 (10 μL/mg tissue) and sonicated on ice. The mixture was centrifuged at 10000 rpm at 4°C for 10 min to precipitate the protein. The concentrations of dopamine and its metabolites HVA and DOPAC were determined using chromatography (ESA, Chelmsford, Ma, USA). S12

Western blotting
The harvested midbrain and striatum were weighed and lysed with RIPA buffer in the presence of protease/phosphatase inhibitors (ratio of tissue to lysate was 1:9, w/v). Equivalent protein samples (30 μg) were then separated on an SDS-PAGE gel (10% for TH detection, 12% for α-syn detection) and transferred onto PVDF membranes. After blocking in 5% bovine serum albumin (BSA) for 2 h, the membranes were incubated with the following primary antibodies at 4°C overnight: TH (1:5000), α-syn (1:1000), and β-actin (1:3000). The blots were rinsed thrice and then incubated with the appropriate secondary antibodies for 2 h. The signals were detected using the ECL Chemiluminescent Reagent. Protein bands were analyzed using ImageJ software with β-actin as the reference standard.

In vivo biocompatibility
After the treatment period, blood was obtained from mice in each group for blood cell and biochemical index analysis. The heart, liver, spleen, lungs, and kidneys were collected and weighed to calculate the organ index according to the following formula: Weigh of organ (mg) Organ Index (%) 100% Weigh of body (g) = Moreover, these organs were fixed in 4% PFA and cut into 5 μm-thick sections.
For pathological analysis, the tissue sections were stained with H&E and potential organ damage was evaluated.

Statistical analysis
All data were expressed as the mean ± SD and statistically analyzed using GraphPad Prism 8.0 software. The statistical analyses included a student's t-test, one-way ANOVA, and two-way ANOVA. P < 0.05 was designated as the threshold for statistical significance.              Table S2. Pharmacokinetic parameters in the plasma and brain (n = 4).